Treating erectile dysfunction by Orchis anatolica extract

ABSTRACT

A composition for treating erectile dysfunction in a male mammal comprising an Orchis anatolica root bulb extract. A method of treating erectile dysfunction in a mammal comprises administering a therapeutically effective amount of Orchis anatolica root bulbs alcohol extract in a male mammal.

TECHNICAL FIELD

The present invention relates in general to treatment of erectiledysfunction (“ED”), and more particularly to, treatment of ED in mammalsby administrating an effective amount of Orchis anatolica root bulbsalcohol extract.

BACKGROUND INFORMATION

Penile erection is a complicated physiological process that involves notonly the blood vessel system, but also the endocrine and nervoussystems. Patients suffering from erectile dysfunction (“ED”) aredefinitely on the increase by the reasons of the expanded life span, theincrease of adult diseases, change of diet, and the increase ofindustrial and traffic accidents. In addition, the increase of mentalstress and physical fatigue resulting from complicated modern life couldcontribute to aggravate this manifestation.

The methods for the treatment of ED are diversified, ranging frommedicinal treatment including male hormones, and injection ofvasodilators in the corpus cavernous smooth muscle, to surgicaltreatments, including vascular surgery, and surgical implantation of apenile prosthesis. Medicinal treatments, such as male hormones,yohimbine, apomorphine, and traszodone, are not suitable for thetreatment of a severe form of ED. Such medicines have side effects, andeven their effects on treating such manifestation are in doubt.Diversified agents able to relax the corpus cavernous smooth muscle havealso been used. Such medication includes adrenergic alpha-receptorblockades, cholines, NO (nitric oxide), peptides, prostaglandin,histamines, calcium channel inhibitors, calcium channel openers,nonspecific vasodilators, and more. Although sildenafil has beenintroduced as a primary treatment for ED, innovation of a specificmedicine that can have a reliable reproducibility has not been reportedyet.

Orchis anatolica Boiss, also known as Anatolian Orchid, belongs to theOrchidaceae family. The Orchis anatolica plant occurs in various partsof the eastern Mediterranean region, including mainland Greece, and anarea extending from the southern Aegean islands of Turkey to Iran. Thisplant is found also in diversified and specific locations within thepine forest regions in northern Jordan. Out of this family of plants,several species are considered important and popular for their effect intreating many diseases. In the literature, a study indicated that thereis an effect of Orchis anatolica root ingestion on the sexual motivationand performance of male rats; however, no tests were carried on theeffect of Orchis anatolica root bulb alcoholic extract on the treatmentof erectile dysfunction in male mammals (see, Allouh et al., “Orchisanatolica Root Ingestion Improves Sexual Motivation and Performance inMale Rats,” Journal of Complementary and Integrative Medicine, Vol. 7,Iss. 1, Article 39, 2010). Thus, there are no previous reports in theliterature emphasizing the effect of Orchis anatolica on ED.

SUMMARY

In accordance with embodiments of the present disclosure, an ethanolextract of Orchis anatolica root bulbs induced the penile erection inmale rodents and rabbits. Embodiments of the present disclosure alsoconfirmed that such extract can be effectively used for the productionof a treatment agent to overcome ED, ranging from mild to severe cases.

Embodiments of the present disclosure provide a method of treating ED ina male mammal comprising administrating an Orchis anatolica root bulbsalcohol extract to a mammal in need thereof.

In embodiments of the present disclosure, the alcohol may be chosen frommethanol, ethanol, propanol, or butanol.

In embodiments of the present disclosure, the alcohol is 70% ethanol.

Embodiments of the present disclosure provide a method of relaxingcorpus cavernous smooth muscles in a male mammal comprisingadministrating a Orchis anatolica root bulbs alcohol extract to a mammalin need thereof.

In embodiments of the present disclosure, the alcohol is chosen frommethanol, ethanol, propanol, or butanol.

In embodiments of the present disclosure, the alcohol is 70% ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a flow chart of a method for treating erectiledysfunction in a mammal, configured in accordance with embodiments ofthe present disclosure.

DETAILED DESCRIPTION

Embodiments of the present disclosure provide a treatment of erectiledysfunction in a male mammal, comprising administering a Orchisanatolica root bulbs alcohol extract to a male mammal.

Referring to FIG. 1, aspects of the present disclosure comprise a firststep 101 of producing an extract of root bulbs of Orchis anatolica(e.g., in either a pharmaceutically or nutraceutically acceptable form),as further described herein, and then, in a second step 102,administering a therapeutically effective amount of the extract to amale mammal.

The extract may be produced in a following manner. Orchis anatolicaplant may be harvested, and then the roots separated and air dried. Thedried roots may be ground into powder and then mixed with a liquid (forexample, distilled water or alcohol (e.g., methanol, ethanol, propanol,or butanol)) before being administered by oral gavage, or being preparedinto an otherwise therapeutically effective amount (for example, in apharmaceutically or nutraceutically acceptable form) to facilitate itsingestion.

Orchis anatolica Boiss belongs to the Orchidaceae family and is alsoknown as Anatolian Orchid. Orchis anatolica plants used in embodimentsof the present disclosure occur in various parts of the easternMediterranean region, including mainland Greece, and an area extendingfrom the southern Aegean islands of Turkey to Iran, and in diversifiedspecific locations within the pine forest regions in northern Jordan. Anextract of Orchis anatolica of embodiments of the present disclosure areextracted from roots. Herein, the roots mean all the non-aerialunderground parts of the plant. The extract may be extracted from rootbulbs of Orchis anatolica. The root bulbs are the potato shaped tuberousroots.

An extract may be manufactured as an alcohol extract. Such an alcoholextract may be selected from a group consisting of methanol, ethanol,propanol, and butanol. The concentration of alcohol may be 100%, and maypreferably be 70%.

From the perspective of histology, a remarkable feature of the malepenis is its core of erectile tissue (“ET”) columns, each enclosed byits own dense, fibrous connective tissue capsule, named the tunicaalbuginea. Erectile tissue contains a specialized arrangement ofarteries, shunts, and venous sinusoids within a matrix of connectivetissue and smooth muscle, forming a highly structured criss-crossing ofinterconnected fibers and spaces that are tensed as the cylinder expandsduring an erection. Penile ET contains numerous variably shaped spaces(i.e., vacuoles, vascular spaces) lined with simple squamous epithelium,which are separated from one another by trabeculae containing connectivetissue and specially arranged smooth muscle cells. Arterial branchesirrigating the corpora cavernosa penetrate the walls of the trabeculaeof these ET and form either capillary plexuses, which supply thevascular spaces, or continue as coiled arteries (helical arteries). Thisvascular arrangement forms an important source of blood supply to thevascular spaces during penile erection. Blood flowing in the deep arteryof the penis may flow either into the corpora cavernosa or into anarterio-venous anastomosis, which connects directly with efferent veins,and which is usually dilated. In a flaccid state, almost all the bloodfrom the deep artery passes directly into the dilated arterio-venousshunt. Therefore, minimal amounts of blood pass into the corporacavernosa. Blood that does enter the corpora divides into two routes:the helicine arteries that empty directly into the blood spaces of theerectile tissue, and the nutritive arteries of the trabeculae, which,after breaking up into a capillary network, re-form into small veins,then empty into the cavernous spaces. Cavernous spaces are drained byveins that pierce the tunica albuginea and constitute the efferentvenous return. During an erection, blood flow into the deep artery ofthe penis increases. Concomitantly, the opening of the arterio-venousanastomosis is reduced by active vasoconstriction, resulting in aslightly dilated artery passing through the tunica albuginea into thecavernous body. The helicine arteries then dilate the cavernous spacesand fill them with blood. Blood flow leaving the cavernous body is notreduced. Herein, the vascular supply to this tissue determines theerectile aspect of the penis, whereas its impairment leads to ED.

An erection, on the other hand, is mediated by parasympathetic impulsesthat pass from the sacral portion of the spinal cord through the pelvicnerves to the penis. These parasympathetic nerve fibers, in contrast tomost other parasympathetic fibers, are believed to release nitric oxide(NO) and/or vaso-active peptide in addition to acetylcholine. Nitricoxide (NO) exhibits a relaxant effect on the penile arteries and thetrabecular meshwork of smooth muscle fibers within the penile tissue.Therefore, the inability of the cavernosal smooth muscle to relax innormal condition could be related to either reduction of NO productionand/or synthesis or to damage or dysfunction of the corporal tissue, acondition known as ED.

ED is defined as the inability of a male to attain or maintain anerection long enough to complete the sexual act. This condition affectsover 50% of men over the age of 50 years. With age, penile tissuebecomes dysfunctional following sexual stimulation as a result of aninability of the penile arteries and the corporal smooth muscle cells torelax leading to the development of the ED. Recent data suggest that bythe age of 40, about 40% of men may suffer from ED, and this isincreased to almost 70% by the end of the sixth decade of life, whichcould be mainly vasculo-genic in origin. The success of the drugsildenafil (Viagra®) in improving erections in men suffering from ED isdue to the fact that this drug is a phosphodiesterase (“PDE”) inhibitorthat improves the relaxation of the penile arteries and the corporasmooth muscle tissue by augmenting the effect of the NO.

Although considerable advanced research has been made to find an idealtreatment for ED, a well potent treatment for this problem has not beenidentified. The use of herbal medicine, enriched by information fromplant research, has always played a primary role in the treatment ofthis condition. Herbal medicinal plants with yohimbine and red KoreanPanax Ginseng are examples of traditional plants treatments for ED,whereas their usage is burdened with serious adverse effects. One of thetherapeutic claims for the Panax ginseng is that it enhances sexualfunction in laboratory animals. A study conducted by the SouthernIllinois University School of Medicine in 2002 found that both forms ofAsian and American Ginseng enhance the copulatory performance in malerats and mice. The effect of Ginseng could be either due to changes inhormone secretion with emphasis on the elevation of the testosteroneserum titer, or as a direct effect of its ginsenoside components on thecentral nervous system and through a feedback mechanism on the gonadaltissues. Another study indicated that Panax Ginseng directly can causeendothelium-dependent vasodilatation and relaxation of rabbit penilecorpus cavernosum in vitro enforcing its role in treatment of ED.

When the extract from root bulbs of Orchis anatolica was used to treatcorpus cavernous smooth muscle in the Example disclosed herein, therelaxation of the smooth muscle thereof increased in a dose-dependentlymanner.

The extract of Orchis anatolica according to embodiments of the presentdisclosure may be added to food as it is or together with other food orfood ingredients, and may be formulated by conventional methods. Thereis no limitation in food applicable to the extract of embodiments of thepresent disclosure. A mixing rate for the effective ingredients may bedetermined as a function of the purpose of use (e.g., prevention,improvement, or treatment).

Embodiments of the present disclosure provide a treating agent forerectile dysfunction containing an alcohol extract of root bulbs ofOrchis anatolica as an active ingredient. One or more pharmaceuticallyacceptable carriers can be additionally added to make a pharmaceuticalformulation containing the extract. The carrier can be selected from agroup consisting of saline, buffered saline, water, glycerol, andethanol, but the selection is not limited thereto.

The extract according to embodiments of the present disclosure may beadministered orally, and may be used in general forms of pharmaceuticalformulations. The extract according to embodiments of the presentdisclosure can be prepared for oral administration by mixing withgenerally used fillers, extenders, binders, wetting agents,disintegrating agents, diluents such as surfactant, and/or excipients.

The effective dosage of the extract according to embodiments of thepresent disclosure may be determined according to age, gender, healthcondition, absorption of an active ingredient, inactivation rate,excretion, and other medicines applied together. Embodiments of thepresent disclosure may include pharmaceutical formulations in dosageunits. This means that the formulations may be present in the form ofindividual parts, for example tablets, coated tablets, capsules, pills,suppositories, and/or ampoules, the active compound content of whichcorresponds to a fraction or a multiple of an individual dose.

Solid formulations for oral administration may be in the form oftablets, pills, dusting powders, and capsules. Liquid formulation fororal administrations may be in the form of suspensions, solutions,emulsions, and/or syrups, and the above mentioned formulations maycontain various excipients, such as wetting agents, sweeteners,aromatics, and/or preservatives, in addition to generally used simplediluents, such as water and/or liquid paraffin.

Embodiments of the present disclosure will be further described in thefollowing Example without, however, limiting the same thereto.

EXAMPLE

In vitro dose dependent relaxation effect of Orchis anatolica root bulbsethanol extract on penile tissue suspended in physiological bathsolution after an induced contraction and compared to Sildenafil and redKorean Panax ginseng for the same therapeutic purpose.

Method:

A water-jacketed physiological organ bath provides a stable andadjustable way of organ stability and temperature control. Substratesand other nutrients that are required to sustain tissue function wereprovided via a physiological solution (e.g., Krebs solution). This willallowed the study of evoked tissue responses to pharmacological agentsand/or electrical stimulation.

Tissue Preparation:

Penile tissues from a mature New Zealand rabbit (e.g., 3-3.5 kg) andalbino rats (e.g., 300-350 g) were dissected out and cleaned of theouter adherent connective tissue, and the two corpora cavernosa integraltissues were obtained. Each corpus cavernosum was cut lengthwise into 4pieces measuring about 2 mm thick and 10-12 mm long out of which a 7-8mm long piece was taken from the middle part of each strip by removingthe two unwanted edges of these strips. The strips were kept in a welloxygenated Krebs solution, pre-cooled to 4° C., and placed in arefrigerator until the initiation of the experiment.

Tissue Mounting and Equilibration:

Each erectile tissue (“ET”) was mounted by clipping both of its endswithin the bath. An ET strip was hooked to one clip end, which was tiedto a metal wire (e.g., mounting hook), which was fixed to a bottom of awater-jacketed chamber. The top end of the strip was clipped to a cottonthread and connected to a lever (e.g., arm), which was connected to aHarvard isotonic transducer. The former was connected to a HarvardUniversal Oscillograph for recording the force of contraction to anisotonic transducer. The tissue strip was stretched against a fixedcaliber weight of 2 gm attached to the lever of the isotonic transducer.The free movable lever end was horizontally adjusted receptive to anymovement according to the contraction and relaxation of the ET withinthe path when stimulated. Before each experiment, the transducer wascalibrated two times against this caliber weight. The bath into whichthe tissue was mounted contained 25 ml of oxygenated Krebs solution withcontinuous oxygenation (gas mixture of 95% O₂ and 5% CO₂ and at 37° C.)and left for 60 minutes before starting the experiment.

Extract Preparation:

Certain specifications for collection of Orchis anatolica plants wereadopted so that the intact roots together with the potato-shaped bulbparts were collected and preserved intact. Bulbs were detached fromroots and left to dry at air temperature, and then separated into twocategories: a large rounded sized bulb resembling the small sized potato(male bulb) and an elongated small sized bulb (female or feeding bulb).The round potato shaped bulb was chopped and grinded using an electricalgrinder until a powder was obtained. Each 500 g of dried and grindedorchis roots was then refluxed in (2 L) 70% ethanol at 50° C. for 36hours in a continuous extraction (soxhlet) apparatus. Ethanol extractwas filtered and concentrated under reduced pressure at 50° C. using arotary evaporator. Red Korean Panax Ginseng was obtained from a SouthKorea market as a concentrated powder vacuumed and sealed in metalcontainer packages of 50 g (commercially obtained from KOREAEXPORT COLTD). Powder was dissolved well in distilled water from which differentconcentrations were used. Different doses of both Orchis anatolicaextract and Red Korean Panax Ginseng were used to obtain and determinethe maximum effect of ET response. A sildenafil concentration of 1 mMwas chosen because it is considered to be a supra-maximum dose based onclinical findings that an oral therapeutic dose of 100 mg will rarelyresult in free plasma concentrations of >40 nM.

Physiological Buffer Solution “Krebs Solution Preparation”:

A 1 liter Krebs stock solution was prepared the day before theexperiment containing the following chemicals: NaCl (125 mM), NaHCO₃ (20mM), KCl (4 mM), NaH₂PO₄ (1.8 mM), MgSO₄ (1.8 mM), CaCl₂ (1.8 mM),glucose (10 mM), and HEPES buffer (10 mM) (see Table 1). For a final 1liter Krebs solution, 725 ml ringer and 700 ml Deionized water wereadded. The solution pH was set to 7.4 after gassing with 95% 0 ₂/5% CO₂for 30 minutes before it was used. Krebs solutions were freshly preparedon each experimental day from the stock solutions stored in therefrigerator.

TABLE 1 Krebs Ringer Buffer Gram/Liter Final Concentration NaCl  8.5 g145 mM  KCL 0.37 g 5.0 mM MgCl₂ 0.26 g 1.3 mM (omitted for Cell prep)NaH₂PO₄ 0.15 g 1.2 mM Glucose  1.8 g  10 mM Hepes  4.8 g  20 mM

Final pH adjusted to 7.4 with 1 M NaOH

Drug Preparation:

Orchis anatolica and Red Korean Panax Ginseng selective doses wereprepared by adding different concentrations of each treatment directlyinto the bath chamber containing a 25 ml Krebs solution (a 0.013 gOrchis anatolica represents a dose of 400 mg/kg mice body weight in vivotreatment). Each treatment was added, multiplied according to the trialdose needed, together with Phenylephrine into the bath chambercontaining a penile strip. To prepare different concentrations ofSildenafil (0.001 g=100 mM or 0.002 g=200 mM), these concentrations wereadded to the Krebs solution accordingly when different concentrationswere needed. These Sildenafil concentrations were chosen as it isconsidered to be a supra-maximum dose based on clinical findings that anoral therapeutic dose of 100 mg will rarely result in free plasmaconcentrations of >40 nM.

Phenylephrine (“PE”) Preparation:

In penile arteries, materials like noradrenaline (“NA”), phenylephrine(“PE”, α₁-AR agonist) caused concentration-dependent contractions. A 100ml Krebs solution containing 0.0509 gm Phenylephrine was added into a100 volumetric glass flask to make stock solution, which was keptrefrigerated until used. 10 ml, 25 ml, and 50 ml of Phenylephrine stocksolution were added to 90 ml, 250 ml, and 500 ml Krebs, respectively, toyield a 250 μM concentrated dose.

Measurements of Tissue and Organ Activity “Recordings Parameters”:

Penile tissue contraction was induced by adding L-Phenylephrine to aKrebs solution in the bath, which was indicated by a downward movementof the transducer, which was recorded on a chart paper as an upwardmovement. The distance between the baseline and the highest point on therecorded trace represented the maximum contraction force generated(“MCF”). The relaxation induced by Sildenafil, Orchis anatolica, and/orRed Korean Panax Gensing, moved the lever upwards, representingrelaxation of the tissue, which was recorded and traced as a downwardmovement on the chart. Distance from the maximum point to the lowestpoint on the trace after the induction of relaxation represented amaximum relaxation force (“MRF”) generated in the tissue. During theseexperiments, the downward tracer recordings represented the state ofrelaxation, and they were measured at repeated intervals of 5, 10, 15,20, 25, and 30 minutes. The decrease in the trace was expressed as apercentage of the maximum contraction produced before inducing therelaxation.

Experimental Protocol:

Strips of penile tissues (e.g., 2 mm×2 mm×7 mm) were mounted inwater-jacketed chambers (25 ml, Harvard-USA), and heated to 37° C. withcontinuous oxygenation (95% oxygen and 5% CO₂). Strips were equilibratedin a NaCl Krebs solution for 1 hour before proceeding, then thissolution was replaced with a high K⁺ Krebs solution (K⁺=109 mM/L) placedin the chambers. The isotonic contraction of the strips within thesolution was recorded until it reached a constant steady state, whichtook about 15-20 minutes. The strips were then washed again with anormal Krebs solution for at least two times, and the tissue was allowedto return to a complete relaxation state (in about 30 minutes). Thetissue then was contracted replacing the normal Krebs solution withL-Phenylephrine (250 mM). After reaching the peak force of contraction(approximately 5-10 minutes), the solution was replaced with a Krebssolution containing L-Phenylephrine (250 mM) and SNP (300 mM), or bothSNP (300 mM) and one of the drugs used in the experiment (Sildenafil,Orchis, and Ginseng), which was added in an appropriate concentration(200-600 mM) to the chamber for the time intervals mentioned above.

Results:

Preliminary results indicated that a dose dependent relaxation of peniletissue was reached when Orchis anatolica was added to the Krebs solutiontogether with phenylephrine. Table 2 indicates results obtained afterdifferent treatments that were applied to the bath chamber (containingstrips of penile tissues (e.g., 2 mm×2 mm×7 mm) mounted inwater-jacketed chambers). These results are expressed in percentage ofcontraction (−%) or relaxation (+%) of the penile tissue recorded.

TABLE 2 Time dependent penile tissue contraction (−) and relaxation (+)response to treatment Treatment 5 10 15 20 25 High K⁺ Krebs −3% −5% −6%−6.5%   −6% L- −6.8%   −8.2%   −9.5%   −10%  −10%  Phenylephrine (0.0509gm stock solution) Orchis 10% 16% 18% 19% 20% anatolica (0.026 mg/ 50 mlKrebs) Orchis 34% 36% 38% 42% 42% anatolica (0.052 mg/ 50 ml Krebs)Panax ginseng  5%  6%  7%  9%  9% (0.052 mg/ 50 ml Krebs) Sildenafil 11%12% 24% (0.002 g/ 50 ml Krebs)Summary:

The preliminary results indicated that an Orchis anatolica plant rootbulbs extract in a concentration of 0.026 g/50 ml induced up to 16%relaxation of the smooth muscle of the penile cavernosal tissue after 10minutes exposure. These results were obtained after inducing contractionusing phenylephrine into the bath with erectile tissue. An additional16% relaxation was further noticed in the erectile tissue strips when ahigher dose of Orchis anatolica (0.052 g/50 ml) was used after 10minutes of its application. These measured parameters indicated greatererectile tissue relaxation effects of Orchis anatolica plant whencompared with Sildenafil, Red Korean Panax Ginseng, and the controls.These results also indicated that the relaxation of the erectile tissuecorpus cavernosum is dose dependent with greater affinity when an Orchisanatolica plant root bulbs extract was used. Thus, an ethanol extractionof an Orchis anatolica root plant can play an important role inimproving male erectile function in penile tissue in mammals.

While the present invention has been described in details and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various additions, omissions, and modificationscan be made without departing from the spirit and scope thereof.

The invention claimed is:
 1. A method for treating erectile dysfunctionin a male mammal in need thereof comprising administering atherapeutically effective amount of an Orchis anatolica root bulbextract to the male mammal, wherein the Orchis anatolica root bulbextract is obtained by extracting Orchis anatolica root bulb with 70%ethanol.
 2. The method as recited in claim 1, wherein thetherapeutically effective amount of the Orchis anatolica root bulbextract results in a relaxation of smooth muscle in penile cavernosaltissue.
 3. The method as recited in claim 1, wherein the therapeuticallyeffective amount of the Orchis anatolica root bulb extract results in arelaxation of corpus cavernosum erectile tissue.